Use of monensin to regulate glycosylation of recombinant proteins

ABSTRACT

Methods of modulating the properties of a cell culture expressing a protein of interest are provided. In various embodiments the methods relate to the addition of cell-cycle inhibitors to growing cell cultures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/033,559, now U.S. Pat. No. 11,130,980, filed Apr. 29, 2016, which isa National Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2014/063211, having an international filing dateof Oct. 30, 2014, which claims the benefit of U.S. Provisional PatentApplication No. 61/898,310, filed Oct. 31, 2013, all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to compounds and processes formodulating one or more properties of a recombinant protein produced bycell culture, including mammalian cell cultures such as CHO cellcultures.

BACKGROUND OF THE INVENTION

Glycosylation is a ubiquitous post-translational modification inmammalian cells; both normal human immunoglobulins and therapeuticmonoclonal antibodies (mAbs) produced in Chinese hamster ovary (CHO)cells are glycoproteins. Although the glycoforms of a protein expressedby CHO host cells are largely determined during cell line generation andclone selection, the presence and/or degree of high mannose glycoformcontent can also be affected by cell culture conditions (Pacis et al.,(2011) Biotechnol Bioeng 108, 2348-2358).

Both pharmacokinetic properties and effector functions of therapeuticmAbs can be affected by glycosylation of the constant region. Terminalsugars such as fucose and galactose may affect antibody-dependentcellular cytoxicity (ADCC) and complement-dependent cytoxicity (CDC;Wright, A. and S. L. Morrison, Trends Biotechnol (1997) 15:26-32). Highmannose glycans may increase serum clearance of certain mAbs thuspotentially affecting efficacy (Goetze, et al., (2011) Glycobiology21:949-59). Alternatively, high mannose glycoforms can increase theaffinity of antibodies for Fc gamma III receptor thus increasing ADCCactivity of certain antibodies (Yu, et al. (2012) MAbs 4:475-87). Thusfor each recombinant mAb a certain glycosylation profile that bestsupports the therapeutic potential of the mAb needs to be maintained.

Methods for manipulating high mannose glycoform content of a protein incell culture include changes in media compositions, osmolality, pH,temperature, etc (Yu, et al., supra, Pacis et al., supra, Chee FurngWong et al. (2005) Biotechnol Bioeng 89:164-177; Ahn, et al. (2008)Biotechnol Bioeng 101:1234-44). The effectiveness of these methods isspecific to cell lines, molecule types and media environment and istypically obtained by trial and error. Additionally, these methods tendto also alter antibody productivity, cell culture behavior and otherantibody quality attributes.

Monensin is a sodium-hydrogen inophore capable of integrating intobiological membranes and thus disturbing sodium-hydrogen gradientsacross those membranes. It is widely used as an antibiotic in the cattleand fowl industry and as a tool for studying intracellular vesiculartrafficking in cultured eukaryotic cells. Addition of monensin has beenreported to inhibit secretion of many different proteins from variouscell types (Fukao, H., et al. (1989) 14:673-84; Kuhn, L. J., et al(1986). J Biol Chem 261:3816-25). Monensin also inhibits glycanprocessing by neutralizing the pH of the Golgi thus affecting thefunction of various glycosylation enzymes (Kubo, R. T. and M. L. Pigeon,(1983) Mol Immunol 20:345-8).

It has been observed that the addition of monensin leads to an increasein high mannose glycoforms on a variety of different proteins expressedin various cell systems (Machamer, C. E. and P. Cresswell (1984) ProcNatl Acad Sci USA 81:1287-91; Kousoulas, K. G., et al. (1983)Intervirology 20: 56-60; Chatterjee, S., et al., (1982) J Virol44:1003-12). However, in most of the published reports short termapplication of monensin is used to study its effects on glycanprocessing; prolonged administration of monensin at micromolarconcentrations is toxic to the cells. Also, no studies have evaluatedthe utility of monensin to modulate the high mannose profile oftherapeutic antibodies produced by CHO production cell lines.

There still exists a need to identify a universal mechanism which canincrease high mannose glycoforms (particularly Mannose 5), on mAbswithout compromising CHO production culture performance and antibodyyield. Such a method would benefit the process development oftherapeutic proteins. The invention provides a method that regulateshigh mannose glycoform content by contacting cells expressing atherapeutic protein with monensin.

SUMMARY OF THE INVENTION

The present invention provides a method for regulating the high mannoseglycoform content of a recombinant protein during a mammalian cellculture process comprising establishing a mammalian cell culture in abioreactor, and contacting the cell culture with monensin. Optionally,the invention further comprises a step of harvesting the recombinantprotein produced by the cell culture. In a further embodiment therecombinant protein produced by the cell culture is purified andformulated in a pharmaceutically acceptable formulation.

In a further embodiment the high mannose glycoform content of arecombinant protein is increased compared to that produced by a culturewhere the cells are not contacted with monensin. In one embodiment thehigh mannose glycan species is Mannose 5 (Man5). In another embodiment,the high mannose glycan species is Mannose 6 (Man6), Mannose 7 (Man7),Mannose 8 (including Mannose 8a and 8b; Man8a and 8b, or Mannose 9(Man9). In a further embodiment the high mannose glycan species comprisea mixture of Man5, Man6, Man7, Man8a, Man8b, and/or Man9.

The invention provides a further embodiment in which the high mannoseglycoform content of a recombinant protein is less than 10%. In anotherembodiment, the high mannose glycoform content of a recombinant proteinis greater than or equal to 10%. In a further embodiment, the highmannose glycoform content of a recombinant protein produced by a cellculture that is contacted with monensin is greater than that produced bya cell culture that is not contacted with monensin by one percentagepoint, two percentage points, three percentage points, four percentagepoints or 5 percentage points. In yet another embodiment, high mannoseglycoform content of a recombinant protein produced by a cell culturethat is contacted with monensin is greater than that produced by a cellculture that is not contacted with monensin by a 6, 7, 8, 9, or 10percentage points. In further embodiments high mannose glycoform contentof a recombinant protein produced by a cell culture that is contactedwith monensin is greater than that produced by a cell culture that isnot contacted with monensin by a 12, 15, 17, 20, 22, 25, 27, 30, 32, 35,37 or 40 percentage points. In yet another embodiment, the high mannoseglycoform content of a recombinant protein produced by a cell culturethat is contacted with monensin is greater than that produced by a cellculture that is not contacted with monensin by 50 percentage points ormore (i.e., 60, 70, 80, 90 or 100 percentage points).

In one embodiment, monensin is added to the cell culture in a singlebolus dose to achieve a final concentration. In one embodiment, thefinal concentration of monensin in the medium is 0.1 nM-1000 nM; inanother embodiment, the concentration is 10 nM-800 nM; in another thefinal concentration is 25 nM-750 nM; in yet another embodiment, thefinal concentration is 50 nM-500 nM. Further aspects of the inventioninclude a method for regulating the high mannose glycoform content of arecombinant protein during a mammalian cell culture process by includingmonensin in the cell culture medium at a final concentration of 50 nM,100 nM, 250 nM; 500 nM; or of 750 nM.

One embodiment of the invention provides a method for regulating thehigh mannose glycoform content of a recombinant protein during amammalian cell culture process by feeding the cells with a mediumcontaining monensin, or to which monensin is added, continuously forbetween one and three days. In one embodiment, the monensin is presentin the cell culture (by virtue of being added to the medium or beingadded to the culture along with medium) for approximately one day (20-28hours); for approximately two days (40-56 hours) or approximately threedays (60-84 hours). In a further embodiment, the monensin is present inthe cell culture (by virtue of being added to the medium or being addedto the culture along with medium) for four days, five days, six days,seven days, eight days, nine days 10 days or longer. For additionalembodiments, the monensin is present in the cell culture for the entireduration of the culture process. In these embodiment, the monensin maybe present at a set, selected concentration, or it may be present inincreasing concentration, or in an initial concentration that isincreased to a higher concentration before being decreased again to theoriginal concentration or another, lower concentration, as describedherein.

A further embodiment of the invention provides a method for regulatingthe high mannose glycoform content of a recombinant protein during amammalian cell culture process by contacting the cells with a mediumcontaining monensin, and simultaneously or sequentially adding monensinseparately to the culture. Additional embodiments include the use ofmonensin in a fed-batch culture and the use of monensin in a perfusionculture. In one embodiment, the culture is perfused using alternatingtangential flow (ATF). In one embodiment, the monensin is present in thecell culture by virtue of being added to the culture in the medium at aselected concentration for a selected period of time, as hereindescribed, and the concentration of monensin is increased, at a selectedpoint of time and for a selected period of time, by the addition ofmonensin to the culture separately from, but optionally along with, thefeed medium or the perfusion medium.

The invention further provides for the addition of monensin to the cellculture between three and 15 days after the culture is established. Inone embodiment, monensin is added to the cell culture at day 3, at day4, at day 5; at day 6; at day 7; at day 8; at day 9; at day 10; at day11; or at day 12 after the culture is established. The monensin ismaintained at a concentration as previously described (in oneembodiment, at a final concentration of 25 nM, 50 nM, 100 nM, 250 nM, of500 nM or of 750 nM) for a period of time between one and seven days,and may be added by any of the herein mentioned methods (i.e., inclusionin feed medium, addition separately from feed medium, etc.).

A further embodiment of the invention provides for the addition ofmonensin to the cell culture between one and 15 days before the cellculture is harvested. In yet another embodiment, the monensin is presentfor the entire duration of the cell culture, from day 0 through harvest.In one embodiment, monensin is added to the cell culture one day beforeharvest; two days, three days; four days; five days; six days; sevendays; eight days; nine days; or ten days before harvest. The monensin ismaintained at a concentration as previously described (in oneembodiment, at a final concentration of 25 nM, 50 nM, 100 nM, 250 nM, of500 nM or of 750 nM; in another embodiment, at one of the aforementionedranges) for a period of time between one and three days. In a furtherembodiment, the amount of monensin is increased overtime, to a steadystate or to a concentration from which it is then decreased, asdescribed herein.

In yet another aspect of the invention, the addition of monensin to thecell culture begins between one and 15 days after the culture isestablished, or between three and 15 days after the culture isestablished, and optionally continues until the cell culture isharvested. In one embodiment, monensin is added to the cell culture oneday after the culture is established; two days, three days; four days;five days; six days; seven days; eight days; nine days; or ten daysafter the culture is established. In a further embodiment, monensin isadded to the cell culture 11 days, 12 days; 13 days; 14 days; 15 days;16 days; 17 days; 18 days; 19 days; 20 days; 21 days or 22 days afterestablishment of the culture. As previously described, in oneembodiment, monensin is present for the entire duration of the cellculture, from day 0 through harvest. In another embodiment, the monensinis maintained at a concentration as described above (in one embodiment,at a final concentration of 25 nM, 50 nM, 100 nM, 250 nM, of 500 nM orof 750 nM; in another embodiment, at one of the aforementioned ranges)for a period of time between one and three days. In a furtherembodiment, the amount of monensin is increased over time, to a steadystate or to a concentration from which it is then decreased, asdescribed herein.

In one embodiment of the invention, monensin is added to the cellculture to achieve a constant concentration; in another embodiment, theconcentration of monensin is varied. For one embodiment, theconcentration of monensin in the cell culture may be held at 25 nM forfrom three to five days, then increased (or ramped up) to 50 nM for fromone day through the duration of the culture. In another embodiment, theconcentration of monensin in the cell culture may be held at 25 nM forfrom three to five days, then increased to 50 nM for three to five days,then tapered again to 25 nM for the duration of the culture. Additionalembodiments comprise shorter or longer time periods during which thelevels of monensin are increased, held steady, and optionally decreased,for example, increased over a period of from one to two days, heldsteady for a period of from one to two days, and optionally decreasedfor a period of from one to two days.

The invention further includes varying the concentration of monensinfrom between 25 nM and 100 nM to between 100 nM and 500 nM, andmaintaining the second, higher concentration of monensin for a period offrom one day through the duration of the culture. In another embodiment,the method optionally comprises a tapering step that reduces theconcentration of monensin to between 25 nM and 100 nM (for a period offrom one day through the duration of the culture). The duration of eachstage can be varied, as described, holding the monensin at a selectedlevel for from three to five days at each stage. Longer time periods mayalso be employed, as may other variations such as gradually increasingthe amount of monensin over a time period and holding the monensinconcentration, or decreasing it gradually.

In one embodiment, the monensin is included in the medium, which can bea feed medium or a perfusion medium, at a selected final concentration(i.e., 25 nM, 50 nM, 100 nM, 250 nM, of 500 nM or of 750 nM); in anotherembodiment, the monensin is added to the cell culture along with themedium, in yet another embodiment the monensin is added separately fromthe medium. The monensin is added to the culture at a rate sufficient toachieve and/or maintain a desired final concentration in the culture. Inone embodiment, the monensin is added at a rate of 1/40- 1/60 of therate at which medium is added to the culture, for example by perfusion;in another embodiment, the monensin is added at a rate of 1/50 of therate. In further embodiments, the rate is varied to achieve a desiredconcentration using calculations that are known in the art. The monensincan be added at a rate that is from 1/10 of that of the culture mediumto 1/100 of that of the culture medium.

In combination with any of the embodiments of the invention describedherein, antifoam may also added into the culture vessel as needed.Alternatively or additionally, 1M Sodium Carbonate or another suitablebase is used to maintain pH at the desired setpoint.

As described herein, in one aspect of the invention the cell culture maybe maintained by perfusion. In one embodiment perfusion begins on orabout day 1 to on or about day 9 of the cell culture. In a relatedembodiment perfusion begins on or about day 3 to on or about day 7 ofthe cell culture. In one embodiment perfusion begins when the cells havereached a production phase. In further embodiments of the invention,perfusion is accomplished by alternating tangential flow. In a relatedembodiment the perfusion is accomplished by alternating tangential flowusing an ultrafilter or a microfilter.

A further embodiment of the invention provides continuous perfusion; inyet a further embodiment the rate of perfusion is constant. Oneembodiment of the invention provides perfusion performed at a rate ofless than or equal to 1.0 working volumes per day. In a relatedembodiment perfusion is performed at a rate that increases during theproduction phase from 0.25 working volume per day to 1.0 working volumeper day during the cell culture. In another related embodiment perfusionis performed at a rate that reaches 1.0 working volume per day on day 9to day 11 of the cell culture. In another related embodiment perfusionis performed at a rate that reaches 1.0 working volume per day on day 10of the cell culture.

In one embodiment the cell culture receives bolus cell culture mediafeeds prior to days 3-7 of the culture.

In yet another aspect of the invention, the cell culture is maintainedby fed batch. In one embodiment of a fed batch culture, the culture isfed three times during production. In a further embodiment, the cultureis fed on a day between day two and four, on a day between day 5 and 7,and on a day between day 8 and 10. Another embodiment provides a fedbatch method in which the culture is fed four times during production.In a still further embodiment, the culture is fed on a day between daytwo and four, on a day between day 5 and 6, on a day between day 7 and8, and on a day between day 8 and 10 or later.

In one embodiment of the invention, monensin is added to a fed batchculture along with the feed medium. Thus, monensin may be added three orfour times during the production process, at the times set forthpreviously. The monensin may be added to the medium (i.e., productionmedium) at a concentration designed to achieve a particularconcentration in the culture, or the monensin may be added to theculture separately from, but along with, the feed medium. In anotherembodiment, the monensin is added directly to he culture on a day ordays during which the culture is not being fed (i.e., no additional feedmedium is added). The concentration of the monensin and the amount oftime it is present in the culture is selected according to theaforementioned parameters.

According to one embodiment of the invention, the mammalian cell cultureis established by inoculating the bioreactor with at least 0.5×10⁶ to3.0×10⁶ cells/mL in a serum-free culture media. In an alternate orfurther embodiment the mammalian cell culture is established byinoculating the bioreactor with at least 0.5×10⁶ to 1.5×10⁶ cells/mL ina serum-free culture media.

The invention may further comprise a temperature shift during theculture. In one embodiment the temperature shift is from 36° C. to 31°C. In one embodiment the invention further comprises a temperature shiftfrom 36° C. to 33° C. In a related embodiment the temperature shiftoccurs at the transition between the growth phase and production phase.In a related embodiment the temperature shift occurs during theproduction phase.

In another embodiment the invention further comprises inducing cellgrowth-arrest by L-asparagine starvation followed by perfusion with aserum-free perfusion media having an L-asparagine concentration of 5 mMor less. In another embodiment the invention further comprises inducingcell growth-arrest by perfusion with a serum-free perfusion media havingan L-asparagine concentration of 5 mM or less. In a related embodimentthe concentration of L-asparagine in the serum-free perfusion media isless than or equal to 5 mM. In a related embodiment the concentration ofL-asparagine in the serum-free perfusion media is less than or equal to4.0 mM. In a related embodiment the concentration of L-asparagine in theserum-free perfusion media is less than or equal to 3.0 mM. In a relatedembodiment the concentration of L-asparagine in the serum-free perfusionmedia is less than or equal to 2.0 mM. In a related embodiment theconcentration of L-asparagine in the serum-free perfusion media is lessthan or equal to 1.0 mM. In a related embodiment the concentration ofL-asparagine in the serum-free perfusion media is 0 mM. In a relatedembodiment the L-asparagine concentration of the cell culture media ismonitored prior to and during L-asparagine starvation.

In yet another embodiment the invention comprises that the packed cellvolume during a production phase is less than or equal to 35%. In arelated embodiment the packed cell volume is less than or equal to 35%.In a related embodiment the packed cell volume is less than or equal to30%.

In a related embodiment the viable cell density of the mammalian cellculture at a packed cell volume less than or equal to 35% is 10×10⁶viable cells/ml to 80×10⁶ viable cells/ml. In another embodiment theviable cell density of the mammalian cell culture is 20×10⁶ viablecells/ml to 30×10⁶ viable cells/ml.

In yet another embodiment the bioreactor has a capacity of at least 500L. In yet another embodiment the bioreactor has a capacity of at least500 L to 2000 L. In yet another embodiment the bioreactor has a capacityof at least 1000 L to 2000 L.

In yet another embodiment the mammalian cells are Chinese Hamster Ovary(CHO) cells. In yet another embodiment the recombinant protein isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a recombinant fusion protein, or acytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 4 present the results obtained when evaluating theeffect of monensin on cell culture performance in bioreactors usingalternating tangential (ATF). Monensin concentration in ATF reactors Ra(gray triangle) and Rb (gray circles) was held at 500 nM over the courseof ˜22 hors starting on day 8 and ending on day 9. Cells in the controlreactor (black circles) were grown in the absence of monensin. Viablecell density is illustrated in FIG. 1. FIG. 2 presents the results ofviability analysis. In FIG. 3, the packed cell volume of the monensinand control tanks, monitored on a daily basis throughout the course ofthe experiment, is shown. Daily spent medium samples were also submittedfor titer analysis. Packed cell volume and titer values were used tocalculate packed cell-adjusted titers, which are shown in FIG. 4.

FIGS. 5-8 represent the high mannose profiles of MAb E produced in ATFsin the presence of monensin. As described for FIG. 1-4, monensinconcentration in ATF reactors Ra and Rb was held at 500 nM over thecourse of ˜22 hours starting on day 8 and ending on day 9. Daily spentmedium samples were submitted for analysis of total high mannoseglycans. The total glycan analysis for the spent media samples is shownin FIG. 5 (Ra—gray triangles, Rb—gray circles, control (nomonensin)—black circles). In addition to total high mannose, theindividual higher order mannose species were analyzed; results are shownin FIG. 6 for the ATF control (no monensin) reactor (total high mannose,black circles; Man5 (black diamonds), Man6 (black triangles), Man7(black asterisk), Man8 (black square), Man9 (black line). FIG. 7 depictsthe same analysis for Ra reactor, and FIG. 8 for the Rb reactor.

FIG. 9-12 depict the predicted and measured high mannose levels and therate of high mannose decrease for MAb E produced in ATF reactors withmonensin. Monensin concentration in ATF reactors Ra and Rb was held at500 nM over the course of ˜22 hours starting on day 8 and ending on day9. For time-points when measured high mannose (black bars) wascontinuing to increase (days 9-11), predicted high mannose values (whitebars) were calculated for Ra (FIG. 9) and Rb (FIG. 10) reactors based onthe assumption that all of the produced MAb E antibodies contain highmannose glycans. For days 13 and on when high mannose levels on MAb Estart decreasing, predicted high mannose values were calculated assumingthat none of the newly produced MAb E antibodies contained any highmannose glycans (FIGS. 9 and 10). Fold titer increase (black bars) andfold high mannose decrease (white bars) were also calculated for thistime period for Ra (FIG. 11) and Rb (FIG. 12).

FIGS. 13 and 14 illustrate the calculated monensin concentration in ATFreactors Ra (FIG. 13) and Rb (FIG. 14). Monensin was bolused at 500 nMinto reactors on day 8 and was maintained at that concentration for aperiod of ˜22 hours. Monensin concentration in tanks after thetermination of monensin administration was calculated based on themedium perfusion rates in the reactors (gray circles). Total measuredpercent high mannose is shown in black circles.

DETAILED DESCRIPTION OF THE INVENTION

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness in the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI (International System of Units)accepted form. Numeric ranges recited herein are inclusive of thenumbers defining the range and include and are supportive of eachinteger within the defined range. The methods and techniques describedherein are generally performed according to conventional methods wellknown in the art and as described in various general and more specificreferences that are cited and discussed throughout the presentspecification unless otherwise indicated. See, e.g., Sambrook et al.Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates(1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

The disclosed methods are applicable to adherent culture or suspensioncultures grown in stirred tank reactors (including traditional batch andfed-batch cell cultures, which may but need not comprise a spin filter),perfusion systems (including alternating tangential flow (“ATF”)cultures, acoustic perfusion systems, depth filter perfusion systems,and other systems), hollow fiber bioreactors (HFB, which in some casesmay be employed in perfusion processes) as well as various other cellculture methods (see, e.g., Tao et al., (2003) Biotechnol. Bioeng.82:751-65; Kuystermans & Al-Rubeai, (2011) “Bioreactor Systems forProducing Antibody from Mammalian Cells” in Antibody Expression andProduction, Cell Engineering 7:25-52, Al-Rubeai (ed) Springer; Catapanoet al., (2009) “Bioreactor Design and Scale-Up” in Cell and TissueReaction Engineering: Principles and Practice, Eibl et al. (eds)Springer-Verlag, incorporated herein by reference in their entireties).

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference.What is described in an embodiment of the invention can be combined withother embodiments of the invention.

Definitions

As used herein, the terms “a” and “an” mean one or more unlessspecifically indicated otherwise. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures used in connection with,and techniques of, cell and tissue culture, molecular biology,immunology, microbiology, genetics and protein and nucleic acidchemistry and hybridization described herein are those well known andcommonly used in the art.

The instant disclosure provides methods of modulating the properties ofcell cultures expressing a “protein of interest;” “protein of interest”includes naturally occurring proteins, recombinant proteins, andengineered proteins (e.g., proteins that do not occur in nature andwhich have been designed and/or created by humans). A protein ofinterest can, but need not be, a protein that is known or suspected tobe therapeutically relevant. Particular examples of a protein ofinterest include antigen binding proteins (as described and definedherein), peptibodies (i.e., a molecule comprising peptide(s) fusedeither directly or indirectly to other molecules such as an Fc domain ofan antibody, where the peptide moiety specifically binds to a desiredtarget; the peptide(s) may be fused to either an Fc region or insertedinto an Fc-Loop, or a modified Fc molecule, for example as described inU.S. Patent Application Publication No. US2006/0140934 incorporatedherein by reference in its entirety), fusion proteins (e.g., Fc fusionproteins, wherein a Fc fragment is fused to a protein or peptide),cytokines, growth factors, hormones and other naturally occurringsecreted proteins, as well as mutant forms of naturally occurringproteins.

The term “antigen binding protein” is used in its broadest sense andmeans a protein comprising a portion that binds to an antigen or targetand, optionally, a scaffold or framework portion that allows the antigenbinding portion to adopt a conformation that promotes binding of theantigen binding protein to the antigen. Examples of antigen bindingproteins include a human antibody, a humanized antibody; a chimericantibody; a recombinant antibody; a single chain antibody; a diabody; atriabody; a tetrabody; a Fab fragment; a F(ab′)₂ fragment; an IgDantibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof.The antigen binding protein can comprise, for example, an alternativeprotein scaffold or artificial scaffold with grafted CDRs or CDRderivatives. Such scaffolds include, but are not limited to,antibody-derived scaffolds comprising mutations introduced to, forexample, stabilize the three-dimensional structure of the antigenbinding protein as well as wholly synthetic scaffolds comprising, forexample, a biocompatible polymer. See, e.g., Korndorfer et al., 2003,Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003);Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptideantibody mimetics (“PAMs”) can be used, as well as scaffolds based onantibody mimetics utilizing fibronectin components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

Naturally occurring immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. From N-terminus to C-terminus, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain can be done in accordance withthe definitions of Kabat et al. in Sequences of Proteins ofImmunological Interest, 5^(th) Ed., US Dept. of Health and HumanServices, PHS, NIH, NIH Publication no. 91-3242, (1991). As desired, theCDRs can also be redefined according an alternative nomenclature scheme,such as that of Chothia (see Chothia & Lesk, (1987) J. Mol. Biol.196:901-917; Chothia et al., (1989) Nature 342:878-883 or Honegger &Pluckthun, (2001) J. Mol. Biol. 309:657-670).

In the context of the instant disclosure an antigen binding protein issaid to “specifically bind” or “selectively bind” its target antigenwhen the dissociation constant (K_(D)) is ≤10⁻⁸ M. The antibodyspecifically binds antigen with “high affinity” when the K_(D) is≤5×10⁻⁹ M, and with “very high affinity” when the K_(D) is ≤5×10⁻⁸ M.

The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified. Additionally, the term“antibody” refers to an intact immunoglobulin or to an antigen bindingportion thereof that competes with the intact antibody for specificbinding, unless otherwise specified. Antigen binding portions can beproduced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies and can form an element of a protein ofinterest. Antigen binding portions include, inter alia, Fab, Fab′,F(ab′)₂, Fv, domain antibodies (dAbs), fragments includingcomplementarity determining regions (CDRs), single-chain antibodies(scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, andpolypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding to the polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H)1 domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S. App.Pub. Nos. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958,Ward et al., (1989) Nature 341:544-546).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,Science 242:423-26 (1988) and Huston et al., (1988) Proc. Natl. Acad.Sci. USA 85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (see, e.g.,Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-48; andPoljak et al., (1994) Structure 2:1121-23). If the two polypeptidechains of a diabody are identical, then a diabody resulting from theirpairing will have two identical antigen binding sites. Polypeptidechains having different sequences can be used to make a diabody with twodifferent antigen binding sites. Similarly, tribodies and tetrabodiesare antibodies comprising three and four polypeptide chains,respectively, and forming three and four antigen binding sites,respectively, which can be the same or different.

One or more CDRs can be incorporated into a molecule either covalentlyor noncovalently to make it an antigen binding protein. An antigenbinding protein can incorporate the CDR(s) as part of a largerpolypeptide chain, can covalently link the CDR(s) to another polypeptidechain, or can incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein can have one or more binding sites. If thereis more than one binding site, the binding sites can be identical to oneanother or can be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

For purposes of clarity, and as described herein, it is noted that anantigen binding protein can, but need not, be of human origin (e.g., ahuman antibody), and in some cases will comprise a non-human protein,for example a rat or murine protein, and in other cases an antigenbinding protein can comprise a hybrid of human and non-human proteins(e.g., a humanized antibody).

A protein of interest can comprise a human antibody. The term “humanantibody” includes all antibodies that have one or more variable andconstant regions derived from human immunoglobulin sequences. In oneembodiment, all of the variable and constant domains are derived fromhuman immunoglobulin sequences (a fully human antibody). Such antibodiescan be prepared in a variety of ways, including through the immunizationwith an antigen of interest of a mouse that is genetically modified toexpress antibodies derived from human heavy and/or light chain-encodinggenes, such as a mouse derived from a Xenomouse®, UltiMab™, orVelocimmune® system. Phage-based approaches can also be employed.

Alternatively, a protein of interest can comprise a humanized antibody.A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. Examples of how to make humanized antibodies can befound in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

An “Fc” region, as the term is used herein, comprises two heavy chainfragments comprising the C_(H)2 and C_(H)3 domains of an antibody. Thetwo heavy chain fragments are held together by two or more disulfidebonds and by hydrophobic interactions of the C_(H)3 domains. Proteins ofinterest comprising an Fc region, including antigen binding proteins andFc fusion proteins, form another aspect of the instant disclosure.

A “hemibody” is an immunologically functional immunoglobulin constructcomprising a complete heavy chain, a complete light chain and a secondheavy chain Fc region paired with the Fc region of the complete heavychain. A linker can, but need not, be employed to join the heavy chainFc region and the second heavy chain Fc region. In particularembodiments a hemibody is a monovalent form of an antigen bindingprotein disclosed herein. In other embodiments, pairs of chargedresidues can be employed to associate one Fc region with the second Fcregion. A hemibody can be a protein of interest in the context of theinstant disclosure.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present. A cell culture can comprise one or more host cells.

The term “hybridoma” means a cell or progeny of a cell resulting fromfusion of an immortalized cell and an antibody-producing cell. Theresulting hybridoma is an immortalized cell that produces antibodies.The individual cells used to create the hybridoma can be from anymammalian source, including, but not limited to, hamster, rat, pig,rabbit, sheep, goat, and human. The term also encompasses trioma celllines, which result when progeny of heterohybrid myeloma fusions, whichare the product of a fusion between human cells and a murine myelomacell line, are subsequently fused with a plasma cell. The term is meantto include any immortalized hybrid cell line that produces antibodiessuch as, for example, quadromas (see, e.g., Milstein et al.,(1983)Nature, 537:3053).

The terms “culture” and “cell culture” are used interchangeably andrefer to a cell population that is maintained in a medium underconditions suitable to survival and/or growth of the cell population. Aswill be clear to those of ordinary skill in the art, these terms alsorefer to the combination comprising the cell population and the mediumin which the population is suspended.

The terms “polypeptide” and “protein” (e.g., as used in the context of aprotein of interest or a polypeptide of interest) are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidues is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterms can also encompass amino acid polymers that have been modified,e.g., by the addition of carbohydrate residues to form glycoproteins, orphosphorylated. Polypeptides and proteins can be produced by anaturally-occurring and non-recombinant cell, or polypeptides andproteins can be produced by a genetically-engineered or recombinantcell. Polypeptides and proteins can comprise molecules having the aminoacid sequence of a native protein, or molecules having deletions from,additions to, and/or substitutions of one or more amino acids of thenative sequence.

The terms “polypeptide” and “protein” encompass molecules comprisingonly naturally occurring amino acids, as well as molecules that comprisenon-naturally occurring amino acids. Examples of non-naturally occurringamino acids (which can be substituted for any naturally-occurring aminoacid found in any sequence disclosed herein, as desired) include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is the aminoterminal direction and the right-hand direction is the carboxyl-terminaldirection, in accordance with standard usage and convention.

A non-limiting list of examples of non-naturally occurring amino acidsthat can be inserted into a protein or polypeptide sequence orsubstituted for a wild-type residue in a protein or polypeptide sequenceinclude β-amino acids, homoamino acids, cyclic amino acids and aminoacids with derivatized side chains. Examples include (in the L-form orD-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline(hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline(Nα-MeHoCit), omithine (Orn), Nα-Methylomithine (Nα-MeOrn or NMeOm),sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg orhR),homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Na-MeLor NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ),norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal),3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic),2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe),para-aminophenylalanine (4 AmP or 4-Amino-Phe), 4-guanidinophenylalanine (Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or“K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe),aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine(benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline(hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad),Nα-methyl valine (NMeVal), N-α-methyl leucine (NMeLeu),Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg),cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α, γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), and other similar amino acids, and derivatized forms of anyof those specifically listed.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used. In oneembodiment 500 L to 2000 L bioreactors are used. In one embodiment, 1000L to 2000 L bioreactors are used.

The term “cell culturing medium” (also called “culture medium,” “cellculture media,” “tissue culture media,”) refers to any nutrient solutionused for growing cells, e.g., animal or mammalian cells, and whichgenerally provides at least one or more components from the following:an energy source (usually in the form of a carbohydrate such asglucose); one or more of all essential amino acids, and generally thetwenty basic amino acids, plus cysteine; vitamins and/or other organiccompounds typically required at low concentrations; lipids or free fattyacids; and trace elements, e.g., inorganic compounds or naturallyoccurring elements that are typically required at very lowconcentrations, usually in the micromolar range.

The nutrient solution may optionally be supplemented with additionaloptional components to optimize growth of cells, such as hormones andother growth factors, e.g., insulin, transferrin, epidermal growthfactor, serum, and the like; salts, e.g., calcium, magnesium andphosphate, and buffers, e.g., HEPES; nucleosides and bases, e.g.,adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates,e.g., hydrolyzed animal or plant protein (peptone or peptone mixtures,which can be obtained from animal byproducts, purified gelatin or plantmaterial); antibiotics, e.g., gentamycin; cell protectants orsurfactants such as Pluronic®F68 (also referred to as Lutrol® F68 andKolliphor® P188; nonionic triblock copolymers composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide));polyamines, e.g., putrescine, spermidine and spermine (see e.g., WIPOPublication No. WO 2008/154014) and pyruvate (see e.g. U.S. Pat. No.8,053,238) depending on the requirements of the cells to be culturedand/or the desired cell culture parameters.

Cell culture media include those that are typically employed in and/orare known for use with any cell culture process, such as, but notlimited to, batch, extended batch, fed-batch and/or perfusion orcontinuous culturing of cells.

A “base” (or batch) cell culture medium refers to a cell culture mediumthat is typically used to initiate a cell culture and is sufficientlycomplete to support the cell culture.

A “growth” cell culture medium refers to a cell culture medium that istypically used in cell cultures during a period of exponential growth, a“growth phase”, and is sufficiently complete to support the cell cultureduring this phase. A growth cell culture medium may also containselection agents that confer resistance or survival to selectablemarkers incorporated into the host cell line. Such selection agentsinclude, but are not limited to, geneticin (G4118), neomycin, hygromycinB, puromycin, zeocin, methionine sulfoximine, methotrexate,glutamine-free cell culture medium, cell culture medium lacking glycine,hypoxanthine and thymidine, or thymidine alone.

A “production” cell culture medium refers to a cell culture medium thatis typically used in cell cultures during the transition whenexponential growth is ending and protein production takes over,“transition” and/or “product” phases, and is sufficiently complete tomaintain a desired cell density, viability and/or product titer duringthis phase.

A “perfusion” cell culture medium refers to a cell culture medium thatis typically used in cell cultures that are maintained by perfusion orcontinuous culture methods and is sufficiently complete to support thecell culture during this process. Perfusion cell culture mediumformulations may be richer or more concentrated than base cell culturemedium formulations to accommodate the method used to remove the spentmedium. Perfusion cell culture medium can be used during both the growthand production phases.

Concentrated cell culture medium can contain some or all of thenutrients necessary to maintain the cell culture; in particular,concentrated medium can contain nutrients identified as or known to beconsumed during the course of the production phase of the cell culture.Concentrated medium may be based on just about any cell culture mediaformulation. Such a concentrated feed medium can contain some or all thecomponents of the cell culture medium at, for example, about 2×, 3×, 4×,5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×,600×, 800×, or even about 1000× of their normal amount.

The components used to prepare cell culture medium may be completelymilled into a powder medium formulation; partially milled with liquidsupplements added to the cell culture medium as needed; or added in acompletely liquid form to the cell culture.

Cell cultures can also be supplemented with independent concentratedfeeds of particular nutrients which may be difficult to formulate or arequickly depleted in cell cultures. Such nutrients may be amino acidssuch as tyrosine, cysteine and/or cystine (see e.g., WIPO PublicationNo. 2012/145682). In one embodiment, a concentrated solution of tyrosineis independently fed to a cell culture grown in a cell culture mediumcontaining tyrosine, such that the concentration of tyrosine in the cellculture does not exceed 8 mM. In another embodiment, a concentratedsolution of tyrosine and cystine is independently fed to the cellculture being grown in a cell culture medium lacking tyrosine, cystineor cysteine. The independent feeds can begin prior to or at the start ofthe production phase. The independent feeds can be accomplished by fedbatch to the cell culture medium on the same or different days as theconcentrated feed medium. The independent feeds can also be perfused onthe same or different days as the perfused medium.

“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. Various tissue culture media,including defined culture media, are commercially available, forexample, any one or a combination of the following cell culture mediacan be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's ModifiedEagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium,Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5AMedium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™300 Series (JRH Biosciences, Lenexa, Kans.), among others. Serum-freeversions of such culture media are also available. Cell culture mediamay be supplemented with additional or increased concentrations ofcomponents such as amino acids, salts, sugars, vitamins, hormones,growth factors, buffers, antibiotics, lipids, trace elements and thelike, depending on the requirements of the cells to be cultured and/orthe desired cell culture parameters. The term “bioreactor” means anyvessel useful for the growth of a cell culture. The cell cultures of theinstant disclosure can be grown in a bioreactor, which can be selectedbased on the application of a protein of interest that is produced bycells growing in the bioreactor. A bioreactor can be of any size so longas it is useful for the culturing of cells; typically, a bioreactor issized appropriate to the volume of cell culture being grown inside ofit. Typically, a bioreactor will be at least 1 liter and may be 2, 5,10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000,10,000, 12,000 liters or more, or any volume in between. The internalconditions of the bioreactor, including, but not limited to pH andtemperature, can be controlled during the culturing period. Those ofordinary skill in the art will be aware of, and will be able to select,suitable bioreactors for use in practicing the present invention basedon the relevant considerations.

“Cell density” refers to the number of cells in a given volume ofculture medium. “Viable cell density” refers to the number of live cellsin a given volume of culture medium, as determined by standard viabilityassays (such as trypan blue dye exclusion method).

The term “cell viability” means the ability of cells in culture tosurvive under a given set of culture conditions or experimentalvariations. The term also refers to that portion of cells which arealive at a particular time in relation to the total number of cells,living and dead, in the culture at that time.

“Packed cell volume” (PCV), also referred to as “percent packed cellvolume” (% PCV), is the ratio of the volume occupied by the cells, tothe total volume of cell culture, expressed as a percentage (seeStettler, et al., (2006) Biotechnol Bioeng. December 20:95(6):1228-33).Packed cell volume is a function of cell density and cell diameter;increases in packed cell volume could arise from increases in eithercell density or cell diameter or both. Packed cell volume is a measureof the solid content in the cell culture. Solids are removed duringharvest and downstream purification. More solids mean more effort toseparate the solid material from the desired product during harvest anddownstream purification steps. Also, the desired product can becometrapped in the solids and lost during the harvest process, resulting ina decreased product yield. Since host cells vary in size and cellcultures also contain dead and dying cells and other cellular debris,packed cell volume is a more accurate way to describe the solid contentwithin a cell culture than cell density or viable cell density. Forexample, a 2000 L culture having a cell density of 50×10⁶ cells/ml wouldhave vastly different packed cell volumes depending on the size of thecells. In addition, some cells, when in a growth-arrested state, willincrease in size, so the packed cell volume prior to growth-arrest andpost growth-arrest will likely be different, due to increase in biomassas a result to cell size increase.

“Growth-arrest”, which may also be referred to as “cell growth-arrest”,is the point where cells stop increasing in number or when the cellcycle no longer progresses. Growth-arrest can be monitored bydetermining the viable cell density of a cell culture. Some cells in agrowth-arrested state may increase in size but not number, so the packedcell volume of a growth-arrested culture may increase. Growth-arrest canbe reversed to some extent, if the cells are not in declining health, byadding reversing the conditions that lead to growth arrest.

The term “titer” means the total amount of a polypeptide or protein ofinterest (which may be a naturally occurring or recombinant protein ofinterest) produced by a cell culture in a given amount of medium volume.Titer can be expressed in units of milligrams or micrograms ofpolypeptide or protein per milliliter (or other measure of volume) ofmedium. “Cumulative titer” is the titer produced by the cells during thecourse of the culture, and can be determined, for example, by measuringdaily titers and using those values to calculate the cumulative titer.

The term “fed-batch culture” refers to a form of suspension culture andmeans a method of culturing cells in which additional components areprovided to the culture at a time or times subsequent to the beginningof the culture process. The provided components typically comprisenutritional supplements for the cells which have been depleted duringthe culturing process. Additionally or alternatively, the additionalcomponents may include supplementary components (e.g., a cell-cycleinhibitory compound). A fed-batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified.

The terms “integrated viable cell density” or “IVCD” are usedinterchangeably and mean the average density of viable cells over thecourse of the culture multiplied by the amount of time the culture hasrun.

“Cumulative viable cell density” (CVCD) is calculated by multiplying anaverage viable cell density (VCD) between two time-points with the timeduration between those two time points. CVCD is the area under the curveformed by plotting the VCD versus time.

Description of Cell Culture Process

During recombinant protein production it is desirable to have acontrolled system where cells are grown to a desired density and thenthe physiological state of the cells is switched to a growth-arrested,high productivity state where the cells use energy and substrates toproduce the recombinant protein of interest instead of making morecells. Various methods for accomplishing this goal exist, and includetemperature shifts and amino acid starvation, as wells as use of acell-cycle inhibitor or other molecule that can arrest cell growthwithout causing cell death.

The production of a recombinant protein begins with establishing amammalian cell production culture of cells that express the protein, ina culture plate, flask, tube, bioreactor or other suitable vessel.Smaller production bioreactors are typically used, in one embodiment thebioreactors are 500 L to 2000 L. In another embodiment, 1000 L-2000 Lbioreactors are used. The seed cell density used to inoculate thebioreactor can have a positive impact on the level of recombinantprotein produced. In one embodiment the bioreactor is inoculated with atleast 0.5×10⁶ up to and beyond 3.0×10⁶ viable cells/mL in a serum-freeculture medium. In another embodiment the inoculation is 1.0×10⁶ viablecells/mL.

The mammalian cells then undergo an exponential growth phase. The cellculture can be maintained without supplemental feeding until a desiredcell density is achieved. In one embodiment the cell culture ismaintained for up to three days with or without supplemental feeding. Inanother embodiment the culture can be inoculated at a desired celldensity to begin the production phase without a brief growth phase. Inany of the embodiments herein the switch from the growth phase toproduction phase can also be initiated by any of the afore-mentionedmethods.

At the transition between the growth phase and the production phase, andduring the production phase, the percent packed cell volume (% PCV) isequal to or less than 35%. The desired packed cell volume maintainedduring the production phase is equal to or less than 35%. In oneembodiment the packed cell volume is equal to or less than 30%. Inanother embodiment the packed cell volume is equal to or less than 20%.In yet another embodiment the packed cell volume is equal to or lessthan 15%. In a further embodiment the packed cell volume is equal to orless than 10%.

The desired viable cell density at the transition between the growth andproduction phases and maintained during the production phase van bevarious depending on the projects. It can be decided based on theequivalent packed cell volume from the historical data. In oneembodiment, the viable cell density is at least about 10×10⁶ viablecells/mL to 80×10⁶ viable cells/mL. In one embodiment the viable celldensity is at least about 10×10⁶ viable cells/mL to 70×10⁶ viablecells/mL. In one embodiment the viable cell density is at least about10×10⁶ viable cells/mL to 60×10⁶ viable cells/mL. In one embodiment theviable cell density is at least about 10×10⁶ viable cells/mL to 50×10⁶viable cells/mL. In one embodiment the viable cell density is at leastabout 10×10⁶ viable cells/mL to 40×10⁶ viable cells/mL. In anotherembodiment the viable cell density is at least about 10×10⁶ viablecells/mL to 30×10⁶ viable cells/mL. In another embodiment the viablecell density is at least about 10×10⁶ viable cells/mL to 20×10⁶ viablecells/mL. In another embodiment, the viable cell density is at leastabout 20×10⁶ viable cells/mL to 30×10⁶ viable cells/mL. In anotherembodiment the viable cell density is at least about 20×10⁶ viablecells/mL to at least about 25×10⁶ viable cells/mL, or at least about20×10⁶ viable cells/mL.

Lower packed cell volume during the production phase helps mitigatedissolved oxygen sparging problems that can hinder higher cell densityperfusion cultures. The lower packed cell volume also allows for asmaller media volume which allows for the use of smaller media storagevessels and can be combined with slower flow rates. Lower packed cellvolume also has less impact on harvest and downstream processing,compared to higher cell biomass cultures. All of which reduces the costsassociated with manufacturing recombinant protein therapeutics.

Three methods are typically used in commercial processes for theproduction of recombinant proteins by mammalian cell culture: batchculture, fed-batch culture, and perfusion culture. Batch culture is adiscontinuous method where cells are grown in a fixed volume of culturemedia for a short period of time followed by a full harvest. Culturesgrown using the batch method experience an increase in cell densityuntil a maximum cell density is reached, followed by a decline in viablecell density as the media components are consumed and levels ofmetabolic by-products (such as lactate and ammonia) accumulate. Harvesttypically occurs at the point when the maximum cell density is achieved(typically 5-10×10⁶ cells/mL, depending on media formulation, cell line,etc). The batch process is the simplest culture method, however viablecell density is limited by the nutrient availability and once the cellsare at maximum density, the culture declines and production decreases.There is no ability to extend a production phase because theaccumulation of waste products and nutrient depletion rapidly lead toculture decline, (typically around 3 to 7 days).

Fed-batch culture improves on the batch process by providing bolus orcontinuous media feeds to replenish those media components that havebeen consumed. Since fed-batch cultures receive additional nutrientsthroughout the run, they have the potential to achieve higher celldensities (>10 to 30×10⁶ cells/ml, depending on media formulation, cellline, etc)) and increased product titers, when compared to the batchmethod. Unlike the batch process, a biphasic culture can be created andsustained by manipulating feeding strategies and media formulations todistinguish the period of cell proliferation to achieve a desired celldensity (the growth phase) from the period of suspended or slow cellgrowth (the production phase). As such, fed batch cultures have thepotential to achieve higher product titers compared to batch cultures.Typically a batch method is used during the growth phase and a fed-batchmethod used during the production phase, but a fed-batch feedingstrategy can be used throughout the entire process. However, unlike thebatch process, bioreactor volume is a limiting factor which limits theamount of feed. Also, as with the batch method, metabolic by-productaccumulation will lead to culture decline, which limits the duration ofthe production phase, about 1.5 to 3 weeks. Fed-batch cultures arediscontinuous and harvest typically occurs when metabolic by-productlevels or culture viability reach predetermined levels. When compared toa batch culture, in which no feeding occurs, a fed batch culture canproduce greater amounts of recombinant protein. See e.g. U.S. Pat. No.5,672,502.

Perfusion methods offer potential improvement over the batch andfed-batch methods by adding fresh media and simultaneously removingspent media. Typical large scale commercial cell culture strategiesstrive to reach high cell densities, 60-90(+)×10⁶ cells/mL where almosta third to over one-half of the reactor volume is biomass. Withperfusion culture, extreme cell densities of >1×10⁸ cells/mL have beenachieved and even higher densities are predicted. Typical perfusioncultures begin with a batch culture start-up lasting for a day or twofollowed by continuous, step-wise and/or intermittent addition of freshfeed media to the culture and simultaneous removal of spent media withthe retention of cells and additional high molecular weight compoundssuch as proteins (based on the filter molecular weight cutoff)throughout the growth and production phases of the culture. Variousmethods, such as sedimentation, centrifugation, or filtration, can beused to remove spent media, while maintaining cell density. Perfusionflow rates of a fraction of a working volume per day up to many multipleworking volumes per day have been reported.

An advantage of the perfusion process is that the production culture canbe maintained for longer periods than batch or fed-batch culturemethods. However, increased media preparation, use, storage and disposalare necessary to support a long term perfusion culture, particularlythose with high cell densities, which also need even more nutrients, andall of this drives the production costs even higher, compared to batchand fed batch methods. In addition, higher cell densities can causeproblems during production, such as maintaining dissolved oxygen levelsand problems with increased gassing including supplying more oxygen andremoving more carbon dioxide, which would result in more foaming and theneed for alterations to antifoam strategies; as well as during harvestand downstream processing where the efforts required to remove theexcessive cell material can result in loss of product, negating thebenefit of increased titer due to increased cell mass.

Also provided is a large scale cell culture strategy that combines fedbatch feeding during the growth phase followed by continuous perfusionduring the production phase. The method targets a production phase wherethe cell culture is maintained at a packed cell volume of less than orequal to 35%.

In one embodiment, a fed-batch culture with bolus feeds is used tomaintain a cell culture during the growth phase. Perfusion feeding canthen be used during a production phase. In one embodiment, perfusionbegins when the cells have reached a production phase. In anotherembodiment, perfusion begins on or about day 3 to on or about day 9 ofthe cell culture. In another embodiment perfusion begins on or about day5 to on or about day 7 of the cell culture.

Using bolus feeding during the growth phase allows the cells totransition into the production phase, resulting in less dependence on atemperature shift as a means of initiating and controlling theproduction phase, however a temperature shift of 36° C. to 31° C. cantake place between the growth phase and production phase. In oneembodiment the shift is from 36° C. to 33° C. In another embodiment theinitiation of cell growth-arrest in the fed-batch culture can beinitiated by exposing the fed-batch culture to a cell-cycle inhibitor.In another embodiment the initiation of cell growth-arrest in thefed-batch culture can be achieved by perfusion with a serum freeperfusion medium comprising a cell-cycle inhibitor.

As described herein, the bioreactor can be inoculated with at least0.5×10⁶ up to and beyond 3.0×10⁶ viable cells/mL in a serum-free culturemedium, for example 1.0×10⁶ viable cells/mL.

Perfusion culture is one in which the cell culture receives freshperfusion feed medium while simultaneously removing spent medium.Perfusion can be continuous, step-wise, intermittent, or a combinationof any or all of any of these. Perfusion rates can be less than aworking volume to many working volumes per day. The cells are retainedin the culture and the spent medium that is removed is substantiallyfree of cells or has significantly fewer cells than the culture.Recombinant proteins expressed by the cell culture can also be retainedin the culture. Perfusion can be accomplished by a number of meansincluding centrifugation, sedimentation, or filtration, See e.g. Voisardet al., (2003), Biotechnology and Bioengineering 82:751-65. An exampleof a filtration method is alternating tangential flow filtration.Alternating tangential flow is maintained by pumping medium throughhollow-fiber filter modules. See e.g. U.S. Pat. No. 6,544,424; Furey(2002) Gen. Eng. News. 22 (7), 62-63.

“Perfusion flow rate” is the amount of media that is passed through(added and removed) from a bioreactor, typically expressed as someportion or multiple of the working volume, in a given time. “Workingvolume” refers to the amount of bioreactor volume used for cell culture.In one embodiment the perfusion flow rate is one working volume or lessper day. Perfusion feed medium can be formulated to maximize perfusionnutrient concentration to minimize perfusion rate.

Cell cultures can be supplemented with concentrated feed mediumcontaining components, such as nutrients and amino acids, which areconsumed during the course of the production phase of the cell culture.Concentrated feed medium may be based on just about any cell culturemedia formulation. Such a concentrated feed medium can contain most ofthe components of the cell culture medium at, for example, about 5×, 6×,7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×,800×, or even about 1000× of their normal amount. Concentrated feedmedia are often used in fed batch culture processes.

The method according to the present invention may be used to improve theproduction of recombinant proteins in multiple phase culture processes.In a multiple stage process, cells are cultured in two or more distinctphases. For example cells may be cultured first in one or more growthphases, under environmental conditions that maximize cell proliferationand viability, then transferred to a production phase, under conditionsthat maximize protein production. In a commercial process for productionof a protein by mammalian cells, there are commonly multiple, forexample, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases thatoccur in different culture vessels preceding a final production culture.

The growth and production phases may be preceded by, or separated by,one or more transition phases. In multiple phase processes, the methodaccording to the present invention can be employed at least during thegrowth and production phase of the final production phase of acommercial cell culture, although it may also be employed in a precedinggrowth phase. A production phase can be conducted at large scale. Alarge scale process can be conducted in a volume of at least about 100,500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters.In one embodiment production is conducted in 500 L, 1000 L and/or 2000 Lbioreactors.

A growth phase may occur at a higher temperature than a productionphase. For example, a growth phase may occur at a first temperature fromabout 35° C. to about 38° C., and a production phase may occur at asecond temperature from about 29° C. to about 37° C., optionally fromabout 30° C. to about 36° C. or from about 30° C. to about 34° C. Inaddition, chemical inducers of protein production, such as, for example,caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be addedat the same time as, before, and/or after a temperature shift. Ifinducers are added after a temperature shift, they can be added from onehour to five days after the temperature shift, optionally from one totwo days after the temperature shift. The cell cultures can bemaintained for days or even weeks while the cells produce the desiredprotein(s).

Samples from the cell culture can be monitored and evaluated using anyof the analytical techniques known in the art. A variety of parametersincluding recombinant protein and medium quality and characteristics canbe monitored for the duration of the culture. Samples can be taken andmonitored intermittently at a desirable frequency, including continuousmonitoring, real time or near real time.

Typically the cell cultures that precede the final production culture(N-x to N-1) are used to generate the seed cells that will be used toinoculate the production bioreactor, the N-1 culture. The seed celldensity can have a positive impact on the level of recombinant proteinproduced. Product levels tend to increase with increasing seed density.Improvement in titer is tied not only to higher seed density, but islikely to be influenced by the metabolic and cell cycle state of thecells that are placed into production.

Seed cells can be produced by any culture method. One such method is aperfusion culture using alternating tangential flow filtration. An N-1bioreactor can be run using alternating tangential flow filtration toprovide cells at high density to inoculate a production bioreactor. TheN-1 stage may be used to grow cells to densities of >90×10⁶ cells/mL.The N-1 bioreactor can be used to generate bolus seed cultures or can beused as a rolling seed stock culture that could be maintained to seedmultiple production bioreactors at high seed cell density. The durationof the growth stage of production can range from 7 to 14 days and can bedesigned so as to maintain cells in exponential growth prior toinoculation of the production bioreactor. Perfusion rates, mediumformulation and timing are optimized to grow cells and deliver them tothe production bioreactor in a state that is most conducive tooptimizing their production. Seed cell densities of >15×10⁶ cells/mL canbe achieved for seeding production bioreactors. Higher seed celldensities at inoculation can decrease or even eliminate the time neededto reach a desired production density.

The invention finds particular utility in regulating the presence and/oramount of glycosylation of a recombinant protein. The cell lines (alsoreferred to as “host cells”) used in the invention are geneticallyengineered to express a polypeptide of commercial or scientificinterest. Cell lines are typically derived from a lineage arising from aprimary culture that can be maintained in culture for an unlimited time.Genetically engineering the cell line involves transfecting,transforming or transducing the cells with a recombinant polynucleotidemolecule, and/or otherwise altering (e.g., by homologous recombinationand gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells and/or cell lines to express a polypeptide of interest are wellknown to those of skill in the art; for example, various techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring LaboratoryPress, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990,pp. 15-69.

Animal cell lines are derived from cells whose progenitors were derivedfrom a multi-cellular animal. One type of animal cell line is amammalian cell line. A wide variety of mammalian cell lines suitable forgrowth in culture are available from the American Type CultureCollection (Manassas, Va.) and commercial vendors. Examples of celllines commonly used in the industry include VERO, BHK, HeLa, CV1(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1),PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells arewidely used for the production of complex recombinant proteins, e.g.cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362;McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990),J. Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines becausethe efficient DHFR selectable and amplifiable gene expression systemallows high level recombinant protein expression in these cells (KaufmanR. J. (1990), Meth Enzymol 185:537-566). In addition, these cells areeasy to manipulate as adherent or suspension cultures and exhibitrelatively good genetic stability. CHO cells and proteins recombinantlyexpressed in them have been extensively characterized and have beenapproved for use in clinical commercial manufacturing by regulatoryagencies.

Proteins of Interest The methods of the invention can be used to culturecells that express recombinant proteins of interest. The expressedrecombinant proteins may be secreted into the culture medium from whichthey can be recovered and/or collected. In addition, the proteins can bepurified, or partially purified, from such culture or component (e.g.,from culture medium) using known processes and products available fromcommercial vendors. The purified proteins can then be “formulated”,meaning buffer exchanged, sterilized, bulk-packaged, and/or packaged fora final user. Suitable formulations for pharmaceutical compositionsinclude those described in Remington's Pharmaceutical Sciences, 18th ed.1995, Mack Publishing Company, Easton, Pa.

Examples of polypeptides that can be produced with the methods of theinvention include proteins comprising amino acid sequences identical toor substantially similar to all or part of one of the followingproteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391),erythropoeitin, thrombopocitin, calcitonin, IL-2, angiopoietin-2(Maisonpierre et al. (1997), Science 277(5322): 55-60), ligand forreceptor activator of NF-kappa B (RANKL, WO 01/36637), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-CSF,Australian Patent No. 588819), mast cell growth factor, stem cell growthfactor (U.S. Pat. No. 6,204,363), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_00682; Rüegg andPytela (1995). Gene 160:257-62) growth hormone, insulin, insulinotropin,insulin-like growth factors, parathyroid hormone, interferons includingα-interferons, γ-interferon, and consensus interferons (U.S. Pat. Nos.4,695,623 and 4,897,471), nerve growth factor, brain-derivedneurotrophic factor, synaptotagmin-like proteins (SLP 1-5),neurotrophin-3, glucagon, interleukins, colony stimulating factors,lymrphotoxin-β, leukemia inhibitory factor, and oncostatin-M.Descriptions of proteins that can be produced according to the inventivemethods may be found in, for example, Human Cytokines: Handbook forBasic and Clinical Research, all volumes (Aggarwal and Gutterman, eds.Blackwell Sciences, Cambridge, Mass., 1998); Growth Factors: A PracticalApproach (McKay and Leigh, eds., Oxford University Press Inc., New York,1993); and The Cytokine Handbook, Vols. 1 and 2 (Thompson and Lotzeeds., Academic Press, San Diego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279). Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Pat. Nos. 4,968,607, and 5,767,064), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(U.S. Pat. Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the 17th International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be producedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

Examples of antibodies that can be produced include, but are not limitedto, those that recognize any one or a combination of proteins including,but not limited to, the above-mentioned proteins and/or the followingantigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25,CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β,IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-1 receptor, IL-2 receptor,IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunits,FGL2, PDGF-β and analogs thereof (see U.S. Pat. Nos. 5,272,064 and5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No.6,235,883) VEGF receptor, hepatocyte growth factor, osteoprotegerinligand, interferon gamma, B lymphocyte stimulator (BlyS, also known asBAFF, THANK, TALL-1, and zTNF4; see Do and Chen-Kiang (2002), CytokineGrowth Factor Rev. 13(1): 19-25), C5 complement, IgE, tumor antigenCA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene productthat is expressed in association with lung cancer), HER-2, HER-3, atumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associatedepitopes that are present in elevated levels in the sera of patientswith colon and/or pancreatic cancer, cancer-associated epitopes orproteins expressed on breast, colon, squamous cell, prostate,pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma,or neuroblastoma cells, the necrotic core of a tumor, integrin alpha 4beta 7, the integrin VLA-4, integrins (including integrins comprisingalpha4beta7), TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor Vila-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphlycoccus aureus.

Specific examples of known antibodies which can be produced using themethods of the invention include but are not limited to adalimurnab,bevacizumab, infliximab, abciximab, alemtuzumab, bapineuzumab,basiliximab, belimumab, briakinumab, brodalumab, canakinumab,certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab,germtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, labetuzumab,mapatumumab, matuzumab, mepolizumab, motavizumab, muromonab-CD3,natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomab,palivizumab, panitumumab, pemtumomab, pertuzumab, ramibizumab,rituximab, rovelizumab, tocilizumab, tositumomab, trastuzumab,ustekinunab, vedolizomab, zalutunumab, and zanolimumab.

The invention can also be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fe portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fe portionof an antibody such as etanercept (a p75 TNFR:Fc), abatacept andbelatacept (CTLA4:Fc).

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

EXAMPLES Example 1

The effect of monensin on high mannose glycans in a recombinant CHO cellline producing an antibody (MAb A) that exhibits very low levels of highmannose glycans was assessed in a six day batch assay. Cells werecentrifuged at 1500 rpm for five minutes and seeded at 2×10⁶ cells perml into batch production medium in a 24 deep-well plate at a finalvolume of 3 ml. Stock solutions of monensin (1000×; eBiosciences Inc.,San Diego, Calif.) were prepared in methanol and added to the culturesat different concentrations and at different time points during theassay (0.1 nM to 50 nM on day 1 and 100-500 nM on day 3); methanol wasadded as vehicle control.

Cell culture parameters were analyzed on day 6 and the correspondingspent medium supernatants were evaluated for antibody titer and glycananalysis to assess the effect of monensin on cell growth, viability,titer and glycan profile of the recombinant antibody. Cell density andviability were measured by Guava easyCyte flow cytometer (Milipore,Billerica, Mass.) with the ViaCount application. Cultures were spundown, supernatants were filtered on 0.4 micron filters, and wereanalyzed for titer and glycan distribution.

Antibody titer was measured by loading filtered cell culturesupernatants over a POROS A/20 Protein A column (Applied Biosystems,Carlsbad, Calif.) equilibrated with 20 mM Tris, 150 nM NaCl, pH 7.0buffer. Antibody elution was performed with 220 mM acetic acid, 150 nMNaCl, pH 2.6 buffer at a mobile phase flow rate of 4.0 ml/min. Elutedantibody was detected at a wavelength of 280 nm. Antibody concentrationwas determined based on a standard curve with a reference antibodystandard. For high molecular weight measurement, antibodies werepurified from spent medium supernatants on ATOLL columns (ATOLL-Bio IncUSA, Lawrance, Kans.) and were then analyzed using size exclusionchromatography.

For glycan analysis, Peptide-N-Glycosidase F (PNGAse F)—releasedN-linked glycans from protein A purified antibodies were labeled with2-aminobenzoic acid (2-AA) and separated by HILIC (hydrophilicinteraction liquid chromatography) in-line with a fluorescence detector.The separation was performed using a Waters Acquity UPLC (Waters,Milford, Mass.). In-line mass spectrometry (MS), using an ion trap massspectrometer (LTQ; Thermo Scientific, Waltham, Mass.) in positive mode,was incorporated to accommodate mass determination of species. Glycanswere injected and bound to the column in high organic conditions andwere then eluted with an increasing gradient of an aqueous ammoniumformate buffer. Fast separation times were achieved using a 1.7 microMsmall particle column format (Acquity UPLC BEH Glycan Column, 2.1×100mm; Waters, Milford, Mass.).

Monensin caused a dose dependent increase in high mannose glycans on therecombinant antibody as shown in Table 1. Man5 was the major highmannose species upregulated upon monensin treatment, though there was aslight increase in higher order mannose structures as well. At monensinconcentrations between 0.1 to 10 nM, there were no impacts on highmannose or cell culture parameters. At high concentrations (50 nMincubated for six days and 500 nM incubated for three days), monensincaused large increases in high mannose at the expense of cell growth,viability and titer. However, when administered as a 25 nM bolus on day0 or either a 200 nM or 100 nM bolus on day 3, monensin increased totalhigh mannose glycans on the recombinant antibody anywhere from 6 to30-fold with no negative impact on cell culture parameters. Methanol,which was used as a vehicle control, did not increase high mannoseglycans.

TABLE 1 Levels of various glycans Total Man5 Man6 Man7 Man8a Man8bAverage HM (%) (%) (%) (%) (%) Control 1.25 0.785 0.06 0.43 0.305 0Methanol 0.65 0.655 0 0 0.375 0   50 nM 25.1 15.725 4.64 4.73 0.8 2.02  25 nM 7.65 6.43 0.59 0.59 0.39 0.115   10 nM 1.6 1.485 0.16 0 0.315 0  5 nM 1.35 1.12 0.18 0.27 0.37 0   1 nM 1.05 0.89 0.13 0.04 0.29 0  0.5nM 0.85 0.785 0.07 0 0.3 0  0.1 nM 0.8 0.775 0.09 0 0.31 0  500 nM(d 3)14.3 11.735 1.545 1.045 0.355 0.2  200 nM(d 3) 27.35 18.585 4.975 3.7950.61 1.17  100 nM(d 3) 33.65 19.365 7.475 6.795 0.8 2.635

Cell culture performance was assessed via viable cell density (VCD),viability and titer measurements of harvested samples. Each barrepresents an average result for duplicate cell culture samples. Eachvalue is an average of duplicates.

TABLE 2 Cell culture parameters VCD Viability Titer Average (10⁶c/ml)(%) (g/L) Control 11 79 3.76 Methanol 10 80 3.835   50 nM  5 72 2.025  25 nM 11 85 5.22   10 nM 11 81 3.635   5 nM 11 82 3.45   1 nM 11 824.265  0.5 nM 11 81 4.415  0.1 nM 10 81 3.835  500 nM(d 3)  5 41 3.055 200 nM(d 3) 11 87 4.43  100 nM(d 3) 11 86 3.515

Taken together these results indicated that monensin has a potential tobe used to increase high mannose glycans on recombinant therapeuticantibodies with no negative impacts on product yield.

Example 2

The effect of monensin on various antibody production cell lines wasevaluated in a mock perfusion setting. Mock perfusion assay is a smallscale, plate-based assay that is designed to mimic perfusion conditionsin bioreactors through daily medium exchanges. For a 10-day mockperfusion assay, passaging cultures of various production cell lineswere diluted 1:5 into chemically defined base perfusion medium in a 24deep-well plate at a final volume of 3 ml per well. Mock perfusion wasinitiated on day 3 when the cells were spun down at 1000 rpm for 5minutes and 25% of each spent culture medium was exchanged with theequivalent volume of fresh perfusion media. Subsequent medium exchangepercentages were 40% on days 4-8 and 50% on day 9. Exchangedsupernatants were stored at 4° C. prior to analysis.

Analysis of cell culture parameters was also started on day 3; viablecell density and viability were analyzed using the ViaCount Guava assayas previously described. Glucose was measured daily starting on day 3and was maintained at 12 g/l. Stored supernatants were analyzed forantibody titer as described previously. Samples of supernatant fluidfrom days 6, 8 and 10 were also analyzed for the presence and type ofglycans by HILIC analysis.

The cell lines used included three production cell lines that are knownto generate mAbs with low high mannose glycans (MAb A, MAb B and MAb C)and one production cell line that consistently produces product withhigh levels of high mannose glycans (MAb D). Monensin was added at afinal concentration of 25 nM on day 3 and from then on one set ofduplicate samples underwent daily partial medium exchanges withperfusion medium containing 25 nM monensin (referred to as “ConstantMonensin” in Table 3 below; columns 3 and 4 of the 24-well plate).Another set of duplicate samples received perfusion medium withincreasing doses of monensin (referred to as “Increasing Monensin” inTable 3 below; columns 5 and 6 of the 24-well plate). Equivalent volumesof methanol were added daily to control cultures (columns 1 and 2 of the24-well plate). This scheme is depicted below:

Columns 1 and 2 Columns 3 and 4 Columns 5 and 6 Row 1: Cells Control:Constant monensin: Increasing producing MAb A Perfused at 40% ofPerfused at 40% of monensin: initial volume with initial volume withPerfused at 40% of media containing media containing 25 initial volumewith equal volume of nM monensin on media containing methanol on days 4,days 4, 5,6,7, and 8, varying monensin 5,6,7,and 8, and 50% on 9 and 50%on 9 on days 4 (25 nM); 5- 9 (50 nM; 50% volume perfused on day 9) Rows2-4: Cells Control: Constant monensin: Increasing producing MAb B,Perfused at 40% of Perfused at 40% of monensin: C, D, respectivelyinitial volume with initial volume with Perfused at 40% of mediacontaining media containing 25 initial volume with equal volume of nMmonensin on media containing methanol on days 4, days 4, 5,6,7, and 8,varying monensin 5,6,7, and 8, and 50% on 9 and 50% on 9 on days 4 (25nM), 5 and 6 (50 nM), 7, 8 and 9 (100 nM; 50% volume perfused on day 9)

On day 10, cell pellets were washed once with cold PBS and fixed in 4%paraformaldehyde for 10 minutes on ice. Cells were then washed once inagain in cold PBS and stored at 4° C. until they wereimmunofluorescently stained as described below

Similar to results obtained with six day batch assay, monensin increasedhigh mannose glycans on all four antibody products tested in a dosedependent manner though the magnitude of upregulation was cell linedependent, as shown in Table 3.

TABLE 3 Levels of various glycans Total HM Total HM Total HM (%) (%) (%)Day 6 Day 8 Day 10 MAb A Control 1.5 1.6 1.4 Constant 55.7 19.7 9monensin Increasing 61.9 44.9 23.2 monensin MAb B Control 3.0 2.8 2.8Constant 9.6 4.6 4.0 monensin Increasing 13.2 12.4 14.0 monensin MAb CControl 4.6 4.7 7.7 Constant 49.6 31.1 30.9 monensin Increasing 56.667.6 65.8 monensin MAb D Control 18.4 26.8 32.2 Constant 70.6 80.1 72.6monensin Increasing 83.0 92.5 94.9 monensin

When compared to the control samples, high mannose levels on antibodiescollected on day 10 of the production assay exhibited increases ofanywhere from 1.5 to 15-fold depending on the monensin dose. The levelsof high mannose glycans decreased over time in cultures that weresubjected to partial daily medium exchange, starting on day 3, withperfusion medium containing 25 nM monensin, but were higher than controlcultures at all time points (Table 3, values shown in rows designated“Constant monensin”). This is likely due to increases in cell numberwith time, thus reducing the per cell dose of monensin at later timepoints.

For one of the cell lines, monensin dose in the perfusion medium wasramped up to 50 nM over the course of the production assay run; for theremaining cell-lines, monensin dose in the perfusion medium was rampedup (increased) to 100 nM. As a result of the increasing monensinconcentration, high mannose levels on the antibodies produced by thesecell lines were held steady from early to later time points (Table 3,values shown in rows designated “Increasing monensin”). For the cellline expressing MAb A (evaluated in Example 1), monensin concentrationin the perfusion medium was not increased beyond 50 nM due to thepreviously observed deleterious effects on cell culture parameters. Assuch, and similarly to what was observed with 25 nM addition condition,high mannose levels on antibodies produced by that cell-line decreasedwith time.

Total high mannose values (Total HM column) and the correspondingdistributions into high mannose species from Man5 through Man9 weredetermined for purified antibody samples collected on day 10. Each valueshown in Table 4 represents an average of duplicates.

TABLE 4 Levels of various glycans Total HM Man5 Man6 Man7 Man8a Man8bMan9 (%) (%) (%) (%) (%) (%) (%) MAb A Control 1.4 0.7 0.1 0.1 0.3 0.10.0 Constant 9 5.6 1.2 1.0 0.4 0.6 0.2 Monensin Increasing 23.2 14.2 3.71.9 0.6 1.4 0.4 monensin MAb B Control 2.8 1.3 0.5 0.4 0.4 0.3 0.0Constant 4.0 2.2 0.7 0.5 0.3 0.3 0.0 Monensin Increasing 14.0 7.7 3.02.0 0.3 0.9 0.1 monensin MAb C Control 7.7 4.5 1.3 0.8 0.7 0.3 0.1Constant 30.9 19.5 5.3 3.6 0.7 1.4 0.3 Monensin Increasing 65.8 31.613.7 12.3 1.0 5.9 1.3 monensin MAb D Control 32.2 20.6 4.3 3.8 0.6 2.60.3 Constant 72.6 30.4 14.7 15.3 0.7 10.1 1.4 Monensin Increasing 94.912.8 14.9 27.2 0.8 34.3 5.0 monensin

In most cases monensin elevated the level of high mannose specieswithout changing their relative distribution (i.e. if Man5 was theprimary high mannose form prior to monensin addition, it typicallystayed the predominant form after monensin administration).

The only exception to this effect was seen on one cell line, thatproducing MAb D. The increasing dose of monensin primarily upregulatedMan7 and Man8(b) high mannose glycans on the mAbs produced by this cellline. This cell line has (in these experiments and in the past)consistently produced mAbs with high levels of high mannose glycans evenunder control culture conditions. The difference in the upregulation ofhigh mannose species in the presence of monensin in this cell-line whencompared to the other tested cell lines could reflect a fundamentaldifference in high mannose processing machinery in these cells.

Example 3

Monensin is known to cause gross changes in Golgi architecturecharacterized by swollen and fragmented cisternae. The structure of theGolgi of CHO production cell lines after monensin treatment was analyzedusing a panel of five different commercially available antibodiesagainst various Golgi proteins with a passaging culture of recombinantcells producing MAb A using immunofluorescence microscopy. Only theantibody against GM130, a Golgi matrix protein, showed a Golgi specificstaining pattern.

Next, day 10 control and monensin-treated mock perfusion cultures of MAbA producing cells and MAb C producing cells were subjected toimmunofluorescence microscopy using GM130 antibody. Paraformaldehydefixed cell pellets were permeablized with 0.1% TritonX-100 made in PBS.Pellets were washed with PBSA (0.5% BSA in PBS) and incubated with GM130antibody (BD Biosciences, San Jose, Calif.) diluted 1:50 in PBSA. Cellswere washed thrice with PBSA and incubated with Alexa 488 conjugatedmouse secondary antibody (Invitrogen, Grand Island, N.Y.) diluted 1:1000in PBSA. Nuclear DNA was visualized with DRAQ5 (Invitrogen, GrandIsland, N.Y.). Images were captured using Zeiss 510 microscope (CarlZeiss, Inc., Jena, Germany) with 63× water immersion lens and analyzedusing LSM image browser software.

No morphological difference were observed between control andmonensin-treated MAb A-producing cells. However, MAb C producing cellstreated with perfusion medium containing continuously increasing amountsof monensin, culminating at 100 nM final monensin concentration in theperfusion medium from days 7-10, showed punctate distribution of GM130protein perhaps indicative of Golgi stress. This kind of change instaining pattern of GM130 has previously been linked to arsenite or heatshock induced cell stress in HeLa cells (Kolobova, E., et al., Exp CellRes, 2009; 315(3) 542-55).

The effects of either constant levels of monensin or increasing levelsof monensin on various cell culture parameters was also evaluated.Viable cell density (VCD) and viability were measured daily starting onday 3. Spent medium samples were collected on days 3-10 and weresubjected to titer analysis. Viable cell densities were used tocalculate cumulative viable cell densities, which were along withcumulative titer values used to calculate specific productivities (qP).Every value shown is an average of duplicates. There was no drop intiter or any other negative cell culture impacts in these cells, despitethe apparent loss of Golgi morphology, as shown in Table 5.

TABLE 5 Cell culture parameters End of Pro- Cumu- CVCD duction lative(10⁶ c- Viability Titer qP day/ml) (%) (g/L) (pg/c/d) MAb A Control 8585 7.5 88 Constant Monensin 57 78 5.9 103 Increasing monensin 57 80 5.8101 MAb B Control 95 85 3.6 38 Constant Monensin 89 85 3.5 40 Increasingmonensin 88 83 3.6 40 MAb C Control 192 69 7.3 38 Constant Monensin 21577 7.4 34 Increasing monensin 208 77 7.1 34 MAb D Control 129 68 5.2 40Constant Monensin 104 68 4.5 43 Increasing monensin 89 53 3.6 41

The effect of monensin on cell culture parameters under mock perfusionconditions was cell line specific, with MAb A cells exhibiting adecrease in total cell mass accumulation followed by a similar, thoughnot as pronounced, negative growth impact on MAb D cells. Monensin hadno effect on the growth or viability of MAb B cells and slightlyincreased cumulative viable cell density and improved the viability ofMAb C cells.

Monensin has a different effect on cell growth and viability dependingon production cell line in question. Monensin has been reported to causeG1/S or G2/M cell cycle block and induce apoptosis in certain lymphomaand renal cancer cells lines. It was shown to decrease the level ofseveral cell cycle related proteins like CDK2, CD6, cyclin A andcyclinB1 and to increase the levels of cell cycle inhibitors p21 and p27(Park, W. H., et al., Int J Oncol. 2003, 22(4): 855-60; Park, W. H., etal., Int J Oncol. 2003, 23(1): 197-204; Park, W. H., et al., Br JHaematol. 2002, 119(2): p. 400-7). The effect of monensin on these cellcycle proteins could explain the negative effect of monensin on MAb Aand MAb D cell growth and viability.

On the other hand, low doses of monensin have been reported to improvecell culture parameters by increasing intracellular Na⁺ levels whichcould explain the improved cell culture performance of MAb C cells inthe presence of monensin (Tenaglia, A. N., C. G. Fry, and G. Van Zant,Exp Hematol. 1985. 13(6): 512-519). Why different production cell-linesrespond differently to monensin is not known and could in part beexplained by the heterogeneity of the cells from which these clonal celllines were derived.

The effect of monensin on titer and specific productivity was also cellline specific (FIG. 3). MAb A cells showed decreased titer but increasedspecific productivity whereas MAb C cells showed increased titer butslightly lower specific productivity in the presence of monensin.Monensin had no impact on either titer or specific productivity of MAb Bcells and slightly decreased the titer of MAb D cells but had no effecton specific productivity. This effect on titer and specific productivitylikely reflects the effect monensin has on cell growth and viability. Ingeneral however, the use of monensin will facilitate large increases inhigh mannose glycans without significant effects on titer or specificproductivity, and virtually no changes in high molecular weight profilesof antibodies produced.

Example 4

The applicability of using monensin to modulate high mannose levels in alarge scale, controlled production setting was evaluated using arecombinant cell line producing MAb E in alternating tangential flow(ATF) bioreactors. MAb E cells grown in growth medium for the seed trainwere used to inoculate the N-1 bioreactor at 6×10⁵ cells/ml in growthmedium. The cells from N-1 bioreactor were then used to inoculate three,2 L production bioreactors (N) at 7.5×10⁵ cells/ml in base perfusionmedium (referred to as control, Ra, and Rb bioreactors).

The production bioreactors were grown for 20 days at pH 7.00, 36° C.,30% DO and 400 rpm agitation. These production tanks were run with theATF system starting on day 3 with 0.5 vol/day perfusion rate, which wasincreased to 0.6 vol/day, 0.8352 vol/day and 1 vol/day on day 6, 7 and8, respectively. Glucose levels were maintained separately at 5 g/Lsince the perfusion medium was prepared without glucose.

Monensin (25 microM stock solution) was added as a single bolus dose toachieve a final concentration of 500 nM into two tanks (Ra and Rb) onday 8; the third tank served as a control tank. Thereafter monensin wasfed continuously for roughly 22 hours at a rate of 1/50 of the perfusionmedium rate to maintain 500 nM concentration in the tanks. Antifoam wasalso added into the tank as needed, while 1M Sodium Carbonate was usedto maintain pH at the desired setpoint. Daily tank samples werecollected for the measurement of various cell culture parameters as wellas for the titer and high mannose analyses.

Sixty micrograms of daily MAb E samples collected from ATF bioreactorswere digested into the Fc/2 and Fab′2 with 60 units of the IdeS enzyme(fabRICATOR, Genovis, Lund, Sweden) in 50 mM Sodium Phosphate, 150 mMNaCl, pH 6.6 with incubation in a 37° C. water bath for 30 minutes. Thedigested samples were then reduced in 4M Guanidine Hydrochloride 50 mMTris, pH 8.3 with 50 mM DTT followed by incubation in a 55° C. heatblock for 10 minutes resulting in reduction to Fc/2, LC and Fd.

Following digestion and reduction, samples were analyzed immediately byRP-HPLC/MS.

Analysis was performed using Waters Acquity Ultra-Performance liquidchromatography (UPLC) system (Waters, Milford, Mass.) coupled to anAgilent MST Time of Flight (TOF) mass spectrometer (AgilentTechnologies, Santa Clara, Calif.). The digested and reduced sampleswere separated on a reversed-phased Waters BEH Phenyl column (1.7 micronparticle size, 2.1×150 mm; Waters, Milford, Mass.) maintained at 80° C.The mobile phases employed for separation were 0.1% TFA (Buffer A) andAcetonitrile, 0.1% TFA (Buffer B).

Five micrograms of each sample was injected and eluted at a flow rate of0.5 mL/min with the following gradient: 30% B was held for 2.5 minutesfollowed by a gradient from 30% to 45% B over a duration of 5 minutes,followed by a gradient from 45% to 100% B over 0.5 minutes; B was heldat 100% for 4 minutes, followed by a gradient of 100% to 30% B over 0.1minutes, and then held at 30% B for the remaining 2.9 minutes. The UVelution was also monitored at a wavelength of 220 nm. Mass data wereextracted from the TIC of the FC/2 peak, followed by deconvolution ofthe extracted spectra using Agilent MassHunter deconvolution software.Ion intensities of the deconvoluted peaks were used for quantificationof the glycan species.

As shown in FIGS. 1-4 FIG. 7, monensin addition had a slight negativeimpact on cell growth and viability but these effects were notsignificant enough to cause any negative impact on the productivity ofthe cultures. In fact, monensin-treated tanks showed marginally improvedtiters as compared to the control tank. Importantly, addition ofmonensin led to a 9-10 fold increase in the levels of high mannoseglycans on the recombinant antibodies (FIGS. 5-9). The primary increasewas seen in Man5 species, though at earlier time points (days 9 and 10)other high order mannose species were also upregulated. From day 11onwards, Man5 was almost the exclusive high mannose species present inthe tanks with negligible quantities of other high mannose speciesdetected.

Comparisons of predicted and measured high mannose levels show that theexpression of high mannose containing antibodies peaked on day 10 where89% of the produced antibodies contained high mannose glycans (FIG. 9).Once monensin amounts became negligible in the tanks (based on perfusionmedium flow rate calculations, days 11 and on), the rate of decrease inthe percentage of antibodies with high mannose glycans was proportionalto the rate at which the titer was increasing (FIG. 10). In other words,high levels of high mannose antibodies were diluted out with newlyproduced antibodies containing low level of high mannose.

Overall, the increase in high mannose levels correlated well with theflow rate-based calculated concentrations of monensin in the tanks, withmaximum increase seen on days 9 and 10 when monensin concentrations wereat their highest and tapering down from day 11 onwards (FIGS. 9-12).After the complete flushing out of monensin from the tanks through theprocess of continuous medium perfusion, high mannose levels decreasedfrom an initial spike of 35-50% down to 15-17% on the day of the harvest(FIGS. 13 and 14).

What is claimed is:
 1. A method of regulating the high mannose denosumabglycoform content of a denosumab composition during a mammalian cellculture process, comprising: establishing, in a serum-free culturemedium in a bioreactor, a cell culture comprising genetically engineeredCHO expressing denosumab, maintaining the CHO cells during a productionphase, and adding monensin to said CHO cell culture during theproduction phase at a concentration of about 25 nM to about 500 nM,wherein the addition of monensin increases the high mannose denosumabglycoform content of the denosumab composition.
 2. The method accordingto claim 1, wherein the amount of monensin is about 25 nM to about 200nM.
 3. The method of claim 2, wherein the monensin is added within about0 to about 6 days from the start of the production phase of the cellculture.
 4. The method of claim 3, wherein the monensin is added withinabout 0 to about 3 days from the start of the production phase of thecell culture.
 5. The method of claim 1, wherein the monensin is added tothe cell culture by addition of a perfusion medium containing themonensin, and wherein the concentration of monensin in the cell cultureis maintained from about 25 nM to about 500 nM.
 6. The method of claim4, wherein the concentration of monensin maintained in the cell cultureis from about 100 nM to about 500 nM.
 7. The method of claim 2, furthercomprising the step of purifying denosumab from a mammalian cellculture.
 8. The method of claim 7, wherein the purified denosumab isformulated into a pharmaceutically acceptable formulation.
 9. The methodof claim 4, further comprising the step of purifying denosumab from amammalian cell culture.
 10. The method according to claim 9, wherein thepurified denosumab is formulated into a pharmaceutically acceptableformulation.